Control of HBV replication by antiviral microRNAs transferred by lentiviral vectors for potential cell and gene therapy approaches

Background: Because molecular mechanisms regulating host cell and virus interactions are not fully understood, we further defined roles of antiviral microRNAs (miRNAs) in HBV replication. Methods: We studied small interfering RNA sequences inserted into the miR-30 backbone in cell systems. Antiviral sequences were cloned into lentiviral vectors upstream of a green fluorescent protein reporter. Transduced cells included HepG2 or HepG2 2.2.15 cell lines and hTERT-FHB fetal human liver cells. HBV replication was analysed by several assays. Results: In 2.2.15 cells treated with constructs primarily targeting HBV polymerase and surface antigen or HBV polymerase and X open reading frames, HBV core protein, HBV DNA and HBV RNA expression decreased. This antiviral effect was more pronounced when the two constructs were expressed together. Similarly, antiviral constructs decreased HBV replication in HepG2 cells transduced with adenoviral vector to express HBV. Although antiviral sequences were expressed in hTERT-FH-B cells, these cells were non-permissive for HBV, possibly owing to expression of miRNAs reported to inhibit HBV replication, whereas these miRNAs were absent in HepG2 cells. Expression of antiviral miRNAs did not affect cell viability or proliferation and no deleterious changes were observed in expression of native cellular miRNAs. Moreover, expression of antiviral miRNA did not affect engraftment and survival of transplanted cells in mice. Conclusions: Identification of effective antiviral miRNAs and transfer of suitable constructs by lentiviral vectors will be helpful for pathophysiological studies of host cell-virus interactions. Simultaneously, this will advance potential mechanisms for cell/gene therapy in those afflicted with chronic hepatitis and refractory liver disease.